Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium.

Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7248, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 01/2004; 100(26):16083-8. DOI: 10.1073/pnas.2634339100
Source: PubMed

ABSTRACT Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO3- secretory pathway that was defective in CF: (i). ASL [HCO3-] was higher in NL than CF ASL; (ii). activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii). NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASL is abnormally regulated under basal conditions because of absent CFTR-dependent HCO3- secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

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Available from: Larry G. Johnson, Feb 12, 2015
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    • "First, loss of CFTR impairs HCO 3 – secretion across airway epithelia cultured from humans [4] [5] and pigs with a disrupted CFTR gene [6]; CF pigs spontaneously develop lung disease that mimics human CF [7]. Second, loss of CFTR reduces the pH of airway surface liquid (ASL) in cultured human airway epithelia [5], of secretions from human submucosal glands studied ex vivo [8], and of ASL studied in vivo, ex vivo, and in epithelial cultures from CF pigs [9]. Third, a reduced pH decreases the activity of antimicrobials in ASL in vivo and in vitro, thereby "
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    ABSTRACT: Disrupted HCO3(-) transport and reduced airway surface liquid (ASL) pH in cystic fibrosis (CF) may initiate airway disease. We hypothesized that ASL pH is reduced in neonates with CF. In neonates with and without CF, we measured pH of nasal ASL. We also measured nasal pH in older children and adults. In neonates with CF, nasal ASL (pH5.2±0.3) was more acidic than in non-CF neonates (pH6.4±0.2). In contrast, nasal pH of CF children and adults was similar to values measured in people without CF. At an age when infection, inflammation and airway wall remodeling are minimal, neonates with CF had an acidic nasal ASL compared to babies without CF. The CF:non-CF pH difference disappeared in older individuals, perhaps because secondary manifestations of disease increase ASL pH. These results aid understanding of CF pathogenesis and suggest opportunities for therapeutic intervention and monitoring of disease.
    Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 01/2014; 13(4). DOI:10.1016/j.jcf.2013.12.006 · 3.82 Impact Factor
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    • "<15%), removing exogenous CO 2 /HCO 3 − reduces the volume of secretions by ≥50% (Joo et al. 2002). These changes reflect the activity of serous cells in the acini because fluid composition is not altered during transit through the gland (Joo et al. 2006), although some acidification occurs when it reaches the airway surface (Fischer et al. 2002; Coakley et al. 2003). In this paper we studied secretion by Calu-3, a human adenocarcinoma cell line widely used as a model for airway submucosal gland serous cells (Haws et al. 1994). "
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    ABSTRACT: Anion exchanger type 2 (AE2/SLC4A2) is an electroneutral Cl-/HCO3- exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (Isc). We have studied the role of AE2 in Cl- and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knockdown cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of CFTR by ~70% without affecting the levels of NKCC1 (Na+-K+-2Cl- cotransporter) or NBCe1 (Na+-nHCO3- cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ~70%, and the secreted fluid exhibited elevated pH and [HCO3-] as compared to the control lines. Unstimulated equivalent short-circuit current (Ieq) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both Ieq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl-/ HCO3- exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl- removal was reduced by ~80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl- loading during cAMP-stimulated secretion of Cl- and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.
    The Journal of Physiology 07/2012; 590(21). DOI:10.1113/jphysiol.2012.236919 · 4.54 Impact Factor
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    • "Despite much progress, the mechanisms and relationship between anion transport and fluid secretion remain uncertain in Calu-3 cells and in gland serous cells. HCO 3 − appears to be the only actively secreted anion under I sc conditions, consistent with bumetanide-insensitive fluid secretion by native airway glands (Corrales et al. 1984), yet most studies indicate that the pH of native gland secretions and airway surface liquid is near neutrality or slightly acidic (Kyle et al. 1990; Coakley et al. 2003; Song et al. 2006; reviewed by Fischer & Widdicombe, 2006). "
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    ABSTRACT: Anion and fluid secretion are both defective in cystic fibrosis (CF) however the transport mechanisms are not well understood. In this study, Cl- and HCO3- secretion was measured using genetically matched CFTR-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- transport under these conditions requires catalyzed synthesis of carbonic acid. Cl- was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl- and fluid transport was bumetanide-insensitive, suggesting basolateral Cl- loading by a NKCC1-independent mechanism. Imposing a transepithelial HCO3- gradient across basolaterally-permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange for Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.
    The Journal of Physiology 07/2012; 590(21). DOI:10.1113/jphysiol.2012.236893 · 4.54 Impact Factor
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